JPS5886093A - Biological preparation of amide - Google Patents

Abstract

PURPOSE:To prepare an amide, in high efficiency, by hydrating the corresponding lower aliphatic nitrile by the action of microorganisms belonging to Pseudomonas genus. CONSTITUTION:Microorganisms belonging to Pseudomonas genus and capable of hydrating a lower aliphatic nitrile to the corresponding amide, e.g. Pseudomonas chlororaphis B23 (FERM BP-187) are cultured aerobically at 6-9pH and 20-37 deg.C in the presence of a 2-4C lower aliphatic nitrile. As an alternative method, the nitrile is added to the aqueous dispersion of the microbial cells separated from the cultured liquid of the above microorganisms, and is made to react with the cells at 0-20 deg.C and 6-9pH for 1-30hr.

Description

Detailed Description of the Invention [■] Background Technical Field of the Invention The present invention relates to a method for producing a corresponding amide by hydrating a lower aliphatic nitrile by the action of microorganisms. More specifically, the present invention relates to a method for the biological production of amides that is proprietary to the microorganisms used.

Lower aliphatic amides, such as acrylamide, are produced by hydration of the corresponding nitrile, such as acrylonitrile, and methods have been proposed in which this hydration is carried out by the action of microorganisms (for example, Japanese Patent Laid-Open No. 51-8618
6 and Japanese Patent Publication No. 1-17918).

SUMMARY OF THE INVENTION The present invention relates to a method for producing the corresponding AND by biologically hydrating a lower aliphatic nitrile with a microorganism belonging to the genus Pseudomonas or Acinetobacter.

Therefore, in the biological production method of amides according to the present invention, the microorganism used is Pseudomonas (Pseud- omonas)
Genus or Acinetobacter (Aclnetobac-1)
er) and has the ability to hydrate the nitrile and convert it into the corresponding amide.

Effects As shown in the examples below, the microorganisms used in the present invention have a particularly high ability to produce acrylamide, and once the acrylamide is produced, it cannot be detected.
Acrylamide is accumulated at a high concentration in the hydration reaction product liquid, and therefore acrylamide can be easily recovered.

Furthermore, since the optimum temperature for the hydration reaction is generally as low as 0 to 20°C, it is advantageous in terms of thermoeconomics.

Detailed description of the invention 1. Target nitrile The present invention uses a hydrated nitrile (target nitrile has 2 to 2 carbon atoms)
This is No. 4's Nido Blade. A specific example of such a nitrile is
Examples are acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, n-butyronitrile and isobutyronitrile. Among these, acrylonitrile is representative, and also has good hydration performance.

In the case of using microorganisms for use in the present invention, the target nitriles tend to have 5 or more carbon atoms, or aromatic nitriles tend to be more difficult to hydrate than nitriles with 2 to 4 carbon atoms.

2. Biological Hydration Reaction The microbiological hydration reaction of nitrile according to the present invention is essentially the same as the above-mentioned known method, except that the microorganisms used are specific.

Therefore, in the present invention, "the nitrile is hydrated and converted into the corresponding amide by the action of the microorganism" means that when the microorganism is cultured in the presence of the nitrile, as well as the bacterial cell culture after culturing the microorganism, In the case of contacting with nitrile, both of the following shall be included. It also includes the case where a bacterial cell or an enzyme produced by the bacterial cell is immobilized and used for a reaction. The biological hydration reaction according to the present invention involves tolyl hydrator-1! to the enzyme produced by this microorganism. ? ) K.

The microorganisms used in the present invention can be cultured according to any suitable method. Generally, the medium used is a carbon source such as glucose, maltose, or dextrin, a nitrogen source such as ammonium sulfate, ammonium chloride, a nitrile other than the target nitrile, e.g. isobutyronitrile in the case of hydration of acrylonitrile, yeast extract, malt extract, etc. , peptone, meat extract, and other organic nutritional sources, phosphate, magnesium, potassium, zinc, iron, manganese, and other inorganic nutritional sources, and others are suitable.

p) of the medium (about 46 to 9, preferably about 7 to 8; the culture temperature is adjusted to about 5 to 30 °C, preferably about δ to 30'C, and in the presence of nitrile to hydrate the culture (culture As mentioned above, the concentration of the nitrile in the culture may be about 0.5 to 2 wt. The nitrile is dispersed in water, physiological saline, or other aqueous medium, and the target nitrile is added thereto to cause a hydration reaction.

In this preferred embodiment, nitrile hydration is carried out by heating an aqueous dispersion containing about 0.1 to 5 weight percent of microbial cells and about 0.5 to 2 weight percent of nitrile at a temperature of about 0 to 20°C, preferably θ to About 1s℃, pH 6-9 @ preferably 7-81!
Allow to react for about 1 to 30 hours at 30°C.

2) K that is consumed is replenished continuously or intermittently so that the concentration in the reaction solution is maintained within the above range.
It is desirable to do so. It is preferable to use an appropriate buffer system in order to maintain the pH value within the above range. Although the microorganism used in the present invention has a low ability to further hydrate the produced amide, the pH of the reaction system is excessively high. ! In general, the above range is appropriate for the reaction temperature, but the hydration reaction can be continued for a long time (by supplementing with nitrile).
For example, if the treatment is carried out for 5 hours or more, a temperature of 5° C. or lower is appropriate.

After hydration for a given time, the nitrile becomes the corresponding amide with a conversion of yioo% and accumulates in the reaction solution at high concentrations. It can be done according to the method. For example, if bacterial cells are removed from the reaction solution by centrifugation, colored substances, impurities, etc. are removed by treatment with activated carbon or ion-exchange resin, and then cleaned under reduced pressure, the desired and, e.g. Acrylamide crystals are obtained.

3. [Microorganism used 1) Type and mycological properties of M The microorganism used in the present invention is one that produces the target amide by hydration of the target nitrile belonging to the genus Pseudomonas or Acnetononacter.

An example of a specific strain of such a microorganism is Pseudomonas chlorolahuis B 23 (P, chlor
-oraphis B23) C, hereinafter abbreviated as B23), Pseudomonas sp, Ps l (P, s
p, ps 1) (hereinafter abbreviated as P81),
Acinetobacter-ip, BG 13 and BCl2 (
A, ip, BCl2 and BCl2) (hereinafter abbreviated as BCl2 and BG 16, respectively).

The main mycological properties of these microorganisms are as follows.

2) Deposit These microorganisms have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the numbers below.

Strain Deposit number Deposit mortar B23 Tetsuko Kenso Yoki No. 6220 Showa 11
Monthly date P81 No. 6221
Same as above BG13 Same as above @6222
Same as above BG16 Same as above No. 62
23 Same as above 4. Experimental Examples Example 1 1) Culture 123 strains were cultured under the following conditions.

(1) Medium dext, phosphorus 0.5%, HPO40.2% MgBO, -7H,OO,02% isobutyronitrile 0.2% NaC10.1
% voice 7.0 (2) Culture conditions: ℃ / 3 days 2) Hydration of acrylonitrile The obtained culture solution is used as an enzyme source, and acrylonitrile is added intermittently to hydrate it. I did it.

The reaction was carried out at 0 to 4°C from the time of addition of acrylonitrile, and the addition of acrylonitrile was continued so that the concentration of 1IJ El = ) was 0.4M.

Approximately IQOg/j of acrylamide was produced in an hour after the start of the reaction. The yield of acrylic acid <F was 99%, and the amount of acrylic acid as a by-product was less than 0.4% of the added acrylonitrile.

Single acrylamide was isolated from the reaction solution by freeze-drying the reaction solution, subjecting it to extraction with methanol, and concentrating the methanol extract to precipitate crystals. After recrystallizing the obtained crystals from methanol, the crystals obtained as colorless plate-like crystals were analyzed for melting point, elemental analysis, NM
It was identified as acrylamide from R and IRK.

Example 2 1) Cultivation It was the same as in Example 1.

2) Hydration of acrylonitrile Acrylonitrile was continuously added to a dispersion of resting bacterial cells, which was obtained by separating bacterial cells from the obtained culture solution, washing them with water, and drying them, for hydration.

The reaction was carried out at 0 to 4°C, and the acrylonitrile concentration was 0.
Acrylonitrile was added intermittently to give a concentration of 4M.

At 7.5 hours after the start of the reaction, 400 g/l of acrylamide was produced linearly and the 0 reaction seemed to be proceeding under sufficient pressure, but at this point the reaction solution became viscous and the reaction was stopped. did.

The acrylamide yield was r99%, and the amount of acrylic acid as a by-product was only about 0.7% of the added acrylonitrile.

The acrylamide produced was collected and identified in the same manner as in Example 1.

Example 3 The substrate specificity of nitrile hydratase in strain 823 was investigated using a bacterial cell method.

ill Cultivation of bacterial cells It was the same as in Example 1.

(2) Hydration of nitrile The bacterial cells were separated from the resulting culture solution, washed with water at °C, and reacted under the following conditions.The volume of the reaction solution was 1 ml.

Substrate nitrile 300 μmol Potassium phosphate powder pH 7.0/100 μmol Bacterial cells 10 mg (as dry bacterial cells) Temperature Heating C Reaction time 1 hour The results obtained were as shown in the table below.

Acetonitrile 0.11 31 Propionitrile 0.41 117 Acrylonitrile 0.35 100 Methacrylonitrile
0.08 28n-butyronitrile
0.20 5-fisobutytetranitrile 0.
The activity of producing 1 μmol of acryl-41' per minute by 1 mg of 18 51 single vm1 cells was defined as 1 unit.

Example 4 This example uses BZ3. , P81%BG13 and BG1
The hydration reaction of acrylonitrile was compared for six strains.

Cultivation of 1 ill Cell Each bacterial cell was cultured in the same manner as in Example 1, except that 0.01% yeast extract was further added to the medium of Example 1.

(2) Hydration of acrylonitrile The bacterial cells were separated from the resulting culture solution, washed with water, and reacted under the following conditions, with a total volume of 1 ml of the reaction solution.

Acrylonitrile 300 μmol Potassium Phosphate Notuar 7-A pH 7,0/100 μmol Bacterial body 10 mg (as dry bacterial cell) Temperature 15
C. Reaction time 2 and 4 hours The results obtained were as shown in the table below.

Procedural amendment filed in 1982 by Kazuo Sugi, Director-General of the Japan Patent Office, 1, Indication of the case, Patent Application No. 1114688, filed in 1982, 2, Biological production method of invention titled Arr, 3, amended. Patent applicant 7, subject of amendment @ “Claims” and “Detailed description of the invention” columns of the specification l≦:〉＼8, Contents of amendment (1) Request for % allowance Correct the range column as shown in the attached sheet.

(2) Page 3 of the specification, lines 5 to 6, “Or Acineto 7
Delete sector genus.

(3) Delete [or genus Acinetobacter] on page 3, lines 1g13-14.

(4) Delete "Acineto 7 sector genus" in line 8jj and line 1θ.

(5) “(hereinafter referred to as 112B) on page 8, lines 14-18.
), Pseudomonas... "of"(
(hereinafter abbreviated as B23) and Pseudomonas ip.

There is PO2 (P, sp, PO2) (hereinafter abbreviated as PO2). ”K correction.

).

(7) Same page, line 7 l'-BG13 Same $
6222 Same as above” and line 8 “BG16 Same as above”
Delete "No. 223 fjl".

(8) On page 16, line 7 from the bottom, “This example is B23
, P81, BG 13 and 1000 strains" is corrected to "B23 and P81 strains in this example." (9) Delete the bottom two rows of the table on page 17 (that is, the description regarding BG 13 and BCl2).

1. In the method of hydrating a nitrile having 2 to 4 carbon atoms by the action of a microorganism and converting it to the corresponding AζFK, the microorganism used is Pseudomonas (Pseudomonaa).
) and has the ability to hydrate a skeleton nitrile and convert it into the corresponding amide.

2. The method of claim 1.1, which comprises adding nitrile to the microorganism culture solution and converting it to the corresponding AND.

3. Adding nitrile to the aqueous dispersion of the image separated from the microbial culture solution and converting it to the corresponding amide;
The method described in claim @1.

4. The method according to any one of claims 2 to 3, in which the addition of nitrile is carried out continuously or intermittently.

5. The nitrile is 7-crylonitrile, and the microorganism is Pseudomonas chlorella fuis 123 (II Koken Soyori No. 6).
220), the method according to any one of claims 1 to 4.

Procedural amendment November 9, 1988 Kazuo Wakasugi, Commissioner of the Patent Office1, Indication of the case 1984 Patent Application No. 1846882, Name of the invention Biological production method of amide3, Person making the amendment Case Number of patent applicants related to 1) Hideaki 7, Grasping of "%#V-Claims" and "Detailed Description of the Invention" of the specification subject to amendment - 7ζ Chingren, 8,
Contents of amendment (1) The scope of claims will be amended as shown in the attached sheet.

(2) Lines 4 to 6 of page 12 of the specification are amended as follows.

Shutomonas sp, No. 188, No. 188
Upper Pg I J
Claim 1, carbon number 2)! In this method, the microorganism used is Pseudomonas (Pseudomonas).
A method for biologically producing an amide, which belongs to the genus S) and has the ability to hydrate and convert a nitrile into a corresponding amide.

2. The method according to claim 1, wherein nitrile is added to the culture solution of the microorganism to convert it into the corresponding amide.

3. Adding nitrile to an aqueous dispersion of bacterial cells isolated from a microbial culture solution and converting it into the corresponding amide;
%IFFai! The method described in item 1 of the scope of the request.

4. Addition of nitrile continuously or intermittently, %
The method according to any one of items 2 to 3 of the 1FFitii search range.

5. The nitrile is acrylonitrile, and the microorganism is Pseudomonas chlorolahuis B23 (Feikokenjoyori No. 18).
7) The method according to any one of claims 1 to 4.

Claims (1)

[Scope of Claims] 1. In a method of hydrating a nitrile having 2 to 4 carbon atoms and converting it into the corresponding ado by the action of a microorganism, the microorganism used is Pseudomonas
) or Acinetoba (Ac1netoba)
cter ), and is characterized by having the ability to hydrate cough nitrile and convert it into the corresponding aphenyl PK. Biological production of amides. 2. The method according to claim 1, wherein =) is added to the culture solution of the microorganism to convert it into the corresponding nitrile. 3. The method according to claim 1, wherein (d) an aqueous dispersion of bacterial cells isolated from a culture solution of an organism is added to convert it into the corresponding nitrile. 4, =) Adding IJL continuously or intermittently;
A method according to any one of claims 2 to 3. 5. The nitrile is acrylonitrile, and the microorganism is Pseudomonas rishirorahuis B23 (Feikoken Bacterial Collection No. 62).
20), the method according to any one of claims 1 to 4.